Veterinary Clinical Sciences Publications
Veterinary Clinical Sciences
2-3-2016
Alterations of the Ileal and Colonic Mucosal
Microbiota in Canine Chronic Enteropathies
Eric Cassmann
Iowa State University, cassmann@iastate.edu
Robin White
Iowa State University
Todd Atherly
United States Department of Agriculture, todd.atherly@ars.usda.gov
Chong Wang
Iowa State University, chwang@iastate.edu
Yaxuan Sun
Iowa State University, evansun@iastate.edu
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Authors
Eric Cassmann, Robin White, Todd Atherly, Chong Wang, Yaxuan Sun, Samir Khoda, Curtis L. Mosher, Mark
R. Ackermann, and Albert Jergens
This article is available at Digital Repository @ Iowa State University: http://lib.dr.iastate.edu/vcs_pubs/5
RESEARCH ARTICLE
Alterations of the Ileal and Colonic Mucosal
Microbiota in Canine Chronic Enteropathies
Eric Cassmann1, Robin White1, Todd Atherly2, Chong Wang3, Yaxuan Sun3,
Samir Khoda1, Curtis Mosher4, Mark Ackermann5, Albert Jergens1*
a11111
1 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Iowa State University, Ames,
Iowa, United States of America, 2 USDA-ARS, Ames, Iowa, United States of America, 3 Department of
Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State
University, Ames, Iowa, United States of America, 4 Department of Genetics, Development & Cell Biology,
College of Liberal Arts and Sciences, Iowa State University, Ames, Iowa, United States of America,
5 Department of Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Ames, Iowa,
United States of America
* ajergens@iastate.edu
Abstract
OPEN ACCESS
Citation: Cassmann E, White R, Atherly T, Wang C,
Sun Y, Khoda S, et al. (2016) Alterations of the Ileal
and Colonic Mucosal Microbiota in Canine Chronic
Enteropathies. PLoS ONE 11(2): e0147321.
doi:10.1371/journal.pone.0147321
Editor: Richard E. Isaacson, University of Minnesota,
UNITED STATES
Received: February 27, 2015
Accepted: December 31, 2015
Background
The intestinal microbiota is increasingly linked to the pathogenesis of chronic enteropathies
(CE) in dogs. While imbalances in duodenal and fecal microbial communities have been
associated with mucosal inflammation, relatively little is known about alterations in mucosal
bacteria seen with CE involving the ileum and colon.
Aim
To investigate the composition and spatial organization of mucosal microbiota in dogs with
CE and controls.
Published: February 3, 2016
Copyright: This is an open access article, free of all
copyright, and may be freely reproduced, distributed,
transmitted, modified, built upon, or otherwise used
by anyone for any lawful purpose. The work is made
available under the Creative Commons CC0 public
domain dedication.
Data Availability Statement: All relevant data are
within the paper.
Funding: The authors received no specific funding
for this work.
Competing Interests: The authors have declared
that no competing interests exist.
Abbreviations: FISH, fluorescence in situ
hybridization; IBD, idiopathic inflammatory bowel
disease; GC, granulomatous colitis; CIBDAI, canine
inflammatory bowel disease activity index; GI,
gastrointestinal; 16S rRNA, 16S ribosomal RNA.
Methods
Tissue sections from endoscopic biopsies of the ileum and colon from 19 dogs with inflammatory bowel disease (IBD), 6 dogs with granulomatous colitis (GC), 12 dogs with intestinal
neoplasia, and 15 controls were studied by fluorescence in situ hybridization (FISH) on a
quantifiable basis.
Results
The ileal and colonic mucosa of healthy dogs and dogs with CE is predominantly colonized
by bacteria localized to free and adherent mucus compartments. CE dogs harbored more
(P < 0.05) mucosal bacteria belonging to the Clostridium-coccoides/Eubacterium rectale
group, Bacteroides, Enterobacteriaceae, and Escherichia coli versus controls. Within the
CE group, IBD dogs had increased (P < 0.05) Enterobacteriaceae and E. coli bacteria
attached onto surface epithelia or invading within the intestinal mucosa. Bacterial invasion
with E. coli was observed in the ileal and colonic mucosa of dogs with GC (P < 0.05). Dogs
with intestinal neoplasia had increased (P < 0.05) adherent (total bacteria,
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Enterobacteriaceae, E. coli) and invasive (Enterobacteriaceae, E. coli, and Bacteroides)
bacteria in biopsy specimens. Increased numbers of total bacteria adherent to the colonic
mucosa were associated with clinical disease severity in IBD dogs (P < 0.05).
Conclusion
Pathogenic events in canine CE are associated with different populations of the ileal and
colonic mucosal microbiota.
Introduction
Chronic enteropathies (CE), including idiopathic inflammatory bowel disease (IBD), food
responsive enteropathy (FRE), and alimentary neoplasia are common causes for persistent or
recurrent gastrointestinal (GI) signs in dogs [1–3]. While the exact etiology for these disorders
remains unknown, accumulating evidence suggests a pivotal role for the intestinal microbiota
in disease pathogenesis. Recent studies using 16S rRNA molecular analyses (ie, high throughput [HTP] sequencing and fluorescence in situ hybridization [FISH]) have shown that canine
CE is associated with altered microbial composition characterized by reduced diversity and/or
selective enrichment with individual bacterial species [4–7].
Canine CE may be multifocal (ie, spectrum of patchy to diffuse mucosal involvement) in its
distribution which supports the acquisition of biopsy samples from multiple intestinal regions
[8]. Moreover, separate studies investigating dogs with CE indicate that histopathologic findings of endoscopically-obtained duodenal, ileal, and colonic biopsies may vary significantly
within the same dog [9]. While bacterial 16S rRNA–based methods have shown an association
between altered microbial composition and duodenal inflammation, relatively little is known
about alterations in mucosal bacteria seen with CE involving the ileum and colon [10,11].
The aim of the present study was to utilize FISH techniques to identify and compare the
mucosal microbiota of concurrent endoscopically-obtained ileal and colonic biopsies from
dogs diagnosed with CE. In addition, we examined the potential relationship of mucosal bacteria, clinical disease activity, and histopathology which might be associated with different causes
of canine CE.
Materials and Methods
Ethical animal use
The collection and analysis of intestinal biopsies obtained endoscopically from dogs with CE
were previously approved by the Iowa State University Institutional Animal Care and Use
Committee. Written informed consent was obtained from all owners of dogs enrolled in separate trials (IACUC Log numbers: 1-11-7061-K, 12-11-7269-K).
Canine CE disease groups and controls
Four dog groups were studied with groups 1–3 representing CE dogs while group 4 dogs served
as controls. The diagnostic evaluation in all CE dogs consisted of extensive medical histories
taken over multiple clinical examinations, hematological and serum biochemistry analyses,
urinalysis, fecal examinations for parasites, diagnostic imaging (including abdominal sonography), and histopathologic examination of mucosal biopsy specimens. In some dogs, samples
were additionally collected for a measurement of serum concentration of pancreatic-lipase
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
immunoreactivity (cPLI), trypsin-like immunoreactivity (cTLI), cobalamin, and folate concentrations. None of the CE dogs had received glucocorticoid or antibiotic therapy within 3 weeks
of being diagnosed with CE. Clinical disease activity was calculated for all dogs using the Canine
Inflammatory Bowel Disease Activity Index (CIBDAI) [3].
Group 1 comprised a group of 19 dogs diagnosed with idiopathic IBD according to previously published criteria [1–3]. The enrollment criteria included persistent (> 3 weeks duration)
GI signs, failure to respond to appropriate dietary (elimination diet fed exclusively for at least 3
weeks) and antibiotic (metronidazole or tylosin administered exclusively for 14 days or more)
trials, failure to document other causes for gastroenteritis by thorough diagnostic testing, and
histopathologic evidence of mucosal inflammation observed in endoscopic biopsies.
Group 2 dogs (n = 6) were diagnosed with granulomatous colitis using traditional IBD diagnostic criteria coupled with specialized immunohistochemical (PAS Stain) and/or molecular
(FISH) confirmation of attaching/invasive Esherichia coli bacteria identified within colonic
mucosal biopsy specimens [10,11].
Group 3 dogs (n = 12) were definitively diagnosed with malignant alimentary tract neoplasia based on microscopic evidence of neoplastic cells infiltrating within the lamina propria of
endoscopically-obtained samples of ileal and/or colonic mucosa. Individual tumor types
included adenocarcinoma (n = 9) and lymphosarcoma (n = 3).
Group 4 dogs served as controls and were comprised of 13 young (<2 years old) healthy
laboratory-reared beagles and 2 young mongrel dogs. Each of these dogs was free of GI signs
over several months preceding diagnostic evaluation. Moreover, control dogs were judged to
be healthy on the basis of normal results on physical examination, hematological and serum
biochemical analysis, urinalysis, multiple fecal examinations, and dirofilaria antigen assay.
Intestinal biopsy and histopathologic examination
All dogs had ileoscopy/colonoscopy performed for the collection of distal intestinal mucosal
biopsy specimens. Dogs were prepared for colonoscopy by withholding food overnight and
administering an oral colonic electrolyte lavage solution, twice, at a dosage of 20 ml/kg. One or
two tepid water enemas (20 ml/kg) were performed in the morning prior to endoscopic examination. Prior to endoscopy, the endoscope and biopsy forceps were thoroughly cleaned and
sterilized using an activated aldehyde solution and gas sterilization, respectively. Multiple (12–
15 samples from the colon; 5–7 samples from the ileum) endoscopic biopsies were obtained
and fixed in 10% neutral buffered formalin and then paraffin embedded for use in histopathology, using HE stains, and for FISH. Ileal mucosal biopsies were collected along a 10 cm segment of distal ileum while colonic mucosal biopsies were obtained from each of the ascending,
transverse, and descending colonic regions. Lastly, mass lesions involving the distal descending
colon and/or rectum were biopsied directly.
Histopathologic examination of endoscopic paraffin-embedded tissue sections was performed by a single pathologist (MA) blinded as to each dog’s history and clinical course. Tissues were graded for severity of intestinal mucosal inflammation using simplified WSAVA
histopathologic criteria [12].
Fluorescence in situ hybridization (FISH)
The formalin-fixed embedded histopathological tissue sections were mounted on glass slides
and evaluated by fluorescence in situ hybridization (FISH) as previously described [13–15]. In
brief, paraffin-embedded tissue specimens were deparaffinized using an automated system by
passage through xylene (3 x 10 min), 100% alcohol (2 x 5 min), 95% ethanol (5 min), and
finally 70% ethanol (5 min). The slides were next rapidly transported in deionized water to the
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
DNA testing laboratory where they were air dried prior to hybridization. FISH probes 5’labeled with either Cy-3 or FITC (Life Sciences) were reconstituted with DNAse-free water and
diluted to a working concentration of 5 ng/μL (Table 1).
For total bacterial counts EUB338-FITC was used. For other analyses, specific probes targeting Clostridium, Bacteroides-Prevotella, Enterobacteriaceae, E coli, Lactobacilli, and Helicobacter were labeled with Cy-3 and were applied simultaneously with the universal bacterial probe
Eub338-FITC [16–20]. This battery of probes was selected to identify specific bacterial groups
and individual bacterial species previously shown to be relevant in the pathogenesis of canine
CE [4–7]. Tissue sections were bathed in 30 μL of DNA–probe mix in a hybridization chamber
maintained at 54°C overnight (12 h). Washing was performed using a wash buffer (hybridization buffer without SDS), the slides were rinsed with sterile water, then allowed to air-dry, and
mounted with SlowFade Gold mounting media (Life Technologies, Carlsbad, CA) and 25X251 cover glass (Fisher Scientific, Pittsburgh, PA).
Probe specificity was confirmed in pilot studies by combining the irrelevant probe nonEub338-FITC with Eub338-Cy-3, and through hybridization experiments with pure isolates of
Clostridia, Bacteroides, Enterobacteriaceae, and E. coli to screen for non-selective hybridization. Archived sections of gastric mucosa from a dog diagnosed with Helicobacter infection
was used as a positive control for helicobacter FISH [14].
In situ quantification of mucosal bacteria
The bacteria were visualized by FISH and 4,6-diamidino-2-phenylindole (DAPI) staining
using a 60x Plan Apo oil objective in conjunction with an optional 1.5x multiplier lens on an
Eclipse TE2000-E fluorescence microscope (Nikon Instruments Inc., Melville NY) and photographed with a CoolSnap EZ camera (Photometrics, Tuscon, AZ) controlled by MetaMorph
software (Nashville, TN). Quantification was only performed when the hybridization signals
were strong and could clearly distinguish intact bacteria morphologically by either 2-color
(universal and bacterial specific FISH probe) or 3-color (FISH probes and DAPI stain) identification. A minimum of 4 different endoscopic biopsy specimens/organs were evaluated for their
mucosal bacterial content. Bacterial quantification was performed in 10 representative fields at
a final observed magnification of 600x or 900x. The ten fields included bacteria found within 4
well-defined mucosal compartments: (1) bacteria contained within the mucosa, (2) bacteria
attached to the surface epithelium, (3) bacteria localized within adherent mucus, and (4) bacteria found within free mucus.
Statistics
Data were analyzed using generalized linear mixed models for each of the colon and ileum tissues. A negative binomial distribution was used in the generalized model for the response, the
number of enumerated bacteria. For each analysis, group (control, IBD, GC, or cancer), probe,
mucosal compartment and their interactions were included as a fixed effect, whereas each dog
was treated as a random effect. F-tests were used to test the significance of main effects and
interactions. If significant overall differences were identified among levels of a factor, post-hoc
pairwise comparisons were performed using t-tests with Tukey's adjustment. To test the correlation between mucosal microbiota and inflammatory indices, spearman’s rank correlation
coefficients and the corresponding p values were calculated. The correlations between the summary counts of attaching and invasive bacteria and each of the severe histologic inflammation
and CIBDAI scores were calculated. All analyses were performed in SAS 9.4 (SAS Institute,
Cary NC) with a P-value of < 0.05 considered significant.
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Table 1. Probes used for fluorescence in situ hybridization. Probes used for in situ bacterial identification.
Probe
Sequence (5’ ! 3’)
Target
Reference
Eub338
GCT GCC TCC CGT AGG AGT
Eubacteria
Amann (1990)
Erec482
GCT TCT TAG TCA RGT ACC G
Eubacterium rectale-Clostridium coccoides
Frank (1998)
Ebac1790
CGT GTT TGC ACA GTG CTG
Enterobacteriaceae
Poulsen (1994)
Ec
GCA AAG GTA TTA ACT TTA CTC CC
E. coli and Shigella
McGregor (1996)
Bac303
CCA ATG TGG GGG ACC TT
Bacteroides spp.
Manz (1996)
Hel717
AGG TCG CCT TCG CAA TGA GTA
Helicobacter spp.
Jergens (2010)
Non-Eub338
ACT CCT ACG GGA GGC AGC
Irrelevant probe
Janeczko (2008)
doi:10.1371/journal.pone.0147321.t001
Results
Patient characteristics
Patient demographics of healthy and diseased dogs, including dietary history at the time of
diagnosis, are found in Table 2. The base-line clinical characteristics in CE dogs were similar to
previous reports (Table 3) [1–3]. Dogs with intestinal cancer were oldest while most IBD dogs
were middle-aged (age range 1–14 years). All CE dogs exhibited variable yet chronic GI signs
indicative of colitis (n = 14) or enterocolitis (n = 23). Three CE dogs had mild disease activity
(including 2 dogs with IBD and 1 dog with GC); 9 dogs had moderate disease activity including
5 dogs with IBD, 1 dog with GC, and 3 dogs with neoplasia); and 12 dogs had severe disease
activity (including 11 dogs with IBD and 1 dog with neoplasia) based on CIBDAI scores. Thirteen dogs with a CIBDAI score of 3 exhibited only signs of colitis. Endoscopic mucosal abnormalities involving the ileum and/or colon (ie, increased friability, granularity, and/or erosions)
were observed in 94% of CE dogs. Histopathologic review of intestinal biopsy specimens
showed a 37% and 63% distribution of mild versus moderate-to-severe inflammatory lesions in
dogs with IBD and GC, respectively.
The mean age of the healthy control dogs was 1.8 years (age range 1–3 years). All dogs were
females. There were no abnormalities in the results of physical examination, CBC, serum biochemical analysis, urinalysis, multiple fecal examinations, dirofilarial antigen assay, endoscopic
examination, or histopathologic findings of mucosal biopsies.
Mucosal bacteria
(i) Total bacteria and distribution by organ and dog group. The number of total bacteria
(ie, summed across all 4 mucosal compartments) identified by each probe is displayed in Tables
4 and 5. The number of Eub338-positive bacteria detected in the ileum and colon of CE dogs
was not significantly (P > 0.05) different from healthy dogs (Fig 1). Within the different groups
of CE dogs, the probes specific for Eubacterium rectale (Erec482), Bacteroides (Bac303), and
members of the family Enterobacteriaceae and E. coli (Ebac1790 and Ec, respectively) hybridized significantly (P < 0.05) more total bacteria than these same bacterial sub-populations
observed in healthy dogs. Dogs with intestinal neoplasia showed significantly (P < 0.05)
increased populations of total bacteria hybridizing against probe Ebac1790 in ileal tissues, and
against probes Eub338, Erec482, Ebac1790, and Bac303 in colonic tissues versus other CE dog
groups. Sub-populations of bacteria (Helicobacter spp) which hybridized against probe Hel717
were significantly (P < 0.05) increased in the ileal and colonic tissues of healthy dogs.
(ii) Disease related differences in number and distribution of mucosal microbiota. The
number of bacteria identified by each probe within different mucosal compartments (ie, free
mucus, adherent mucus, attached to surface epithelia, and invasive within mucosa) is displayed
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Table 2. Dog cohort demographics. Canine cohort demographics with regards to age, gender, breed, and diet at the time of endoscopic biopsy.
Cohort
Age
Gender
Breed
Diet
Healthy control
2
FS
Beagle
Purina maintenance ration
Healthy control
1
FS
Beagle
Purina maintenance ration
Healthy control
2
FS
Beagle
Purina maintenance ration
Healthy control
2
FS
Beagle
Purina maintenance ration
Healthy control
1
FS
Beagle
Purina maintenance ration
Healthy control
2
FS
Beagle
Purina maintenance ration
Healthy control
3
FS
Beagle
Purina maintenance ration
Healthy control
2
FS
Beagle
Purina maintenance ration
Healthy control
2
FS
Beagle
Purina maintenance ration
Healthy control
3
FS
Beagle
Purina maintenance ration
Healthy control
2
FS
Beagle
Purina maintenance ration
Healthy control
2
FS
Beagle
Purina maintenance ration
Healthy control
1
FS
Beagle
Purina maintenance ration
Healthy control
1
F
Mongrel
Purina maintenance ration
Healthy control
1
F
Mongrel
Purina maintenance ration
Idiopathic IBD
5
MC
Dachshund
Prescription Hill’s i/d
Idiopathic IBD
9
F
Standard Poodle
Prescription Hill’s z/d
Idiopathic IBD
2
MC
Pembroke Corgi
Prescription Hill’s i/d
Idiopathic IBD
5
MC
Weimarener
IVD Potato and Venison
Idiopathic IBD
4
FS
Miniature Schanauzer
Royal Canin Low fat Duck /Potato
Idiopathic IBD
2
MC
Jack Russel Terrier
Prescription Hill’s Duck/Potato
Idiopathic IBD
6
FS
Springer Spaniel
Prescription Hill’s i/d
Idiopathic IBD
11
FS
Boxer
Natural Balance Duck/Potato
Idiopathic IBD
2
FS
Giant Schnauzer
Purina H/A
Idiopathic IBD
1
MC
Rottweiler
Sojo’s Grain Free Diet
Idiopathic IBD
1
MC
Great Pyrenees
Homemade–Chicken/Rice
Idiopathic IBD
4
FS
Beagle
Prescription Hill’s z/d
Idiopathic IBD
1
MC
Rottweiler
Idiopathic IBD
2
FS
Havanese
Prescription Hill’s z/d
Idiopathic IBD
11
FS
Labrador Retriever
Prescription Hill’s d/d (Venison)
Idiopathic IBD
11
MC
West highland White Terrier
Prescription Hill’s d/d (Venison)
Idiopathic IBD
4
FS
Shih Tzu
Prescription Hill’s Duck/Potato
Idiopathic IBD
5
MC
English Bulldog
Prescription Hill’s z/d
Idiopathic IBD
9
FS
Grey Hound
Prescription Hill’s z/d
Granulomatous colitis
1
FS
Boxer
Nature’s Recipe–Chicken/Barley/Rice
Royal Canin Low fat Duck /Potato
Granulomatous colitis
1
FS
Boxer
Homemade–Chicken/Rice
Granulomatous colitis
1
FS
Boxer
Prescription Hill’s i/d
Granulomatous colitis
1
FS
Boxer
Purina EN Diet
Granulomatous colitis
1
FS
Boxer
Prescription Hill’s z/d
Prescription Hill’s z/d
Granulomatous colitis
1
FS
Boxer
Intestinal AdenoCA
11
MC
Brittany Spaniel
Beneful Dry Ration
Intestinal AdenoCA
8
MC
Shih Tzu
Prescription Hill’s r/d
Intestinal AdenoCA
11
MC
Shetland Sheep dog
Prescription Hill’s i/d
Intestinal AdenoCA
9
MC
Labrador Retriever
Iams Low-Residue
Intestinal AdenoCA
13
MC
Pembroke Corgi
Eagle Pack Holistic Fish
Intestinal AdenoCA
9
MC
Labrador Retriever
Prescription Hill’s i/d
Intestinal AdenoCA
7
FS
German Shorthair Pointer
Prescription Hill’s i/d
(Continued)
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Table 2. (Continued)
Cohort
Age
Gender
Breed
Diet
Intestinal AdenoCA
11
Intestinal AdenoCA
10
MC
Mongrel
Prescription Hill’s z/d
MC
German Shorthair Pointer
Intestinal LSA
2
Prescription Hill’s r/d
MC
Beagle
Prescription Hill’s z/d
Intestinal LSA
Intestinal LSA
9
FS
Mongrel
Prescription Hill’s i/d
6
MC
Boxer
Homemade–Chicken/Rice
doi:10.1371/journal.pone.0147321.t002
in Tables 6 (ileum) and 7 (colon). The number of bacteria hybridizing against probes Eub338,
Erec482, Ebac1790, Ec, and Bac303 was increased (P < 0.05) in the free and adherent mucus
compartments of CE dogs versus healthy dogs. In colonic biopsy specimens, significant
(P < 0.05) differences in bacteria between mucosal compartments of CE dog were observed for
probes hybridizing against total bacteria (Eub338), Eubacterium rectale (Erec482), Enterobacteriaceae (Ebac1790), E. coli (Ec), Bacteroides (Bac303) and Helicobacter spp (Hel717). These
same differences in bacterial numbers between mucosal compartments of CE dogs were
observed in ileal biopsies with the exception of bacteria hybridizing against probe Hel717.
Dogs with cancer had the greatest (P < 0.05) number of mucus-laden bacteria versus IBD dogs
and dogs diagnosed with GC.
The spatial distribution of attaching bacteria in dogs with CE was significantly (P < 0.05)
different from healthy dogs, with higher numbers of total bacteria (Eub338), Eubacterium rectale (Erec482), Enterobacteriaceae (Ebac1790), and E. coli (Ec) detected within colonic tissues
(Fig 2). Some of these mucosal populations were characterized as biofilms of bacteria adherent
to mucosal epithelia (Fig 3). Dogs with colonic neoplasia had increased (P < 0.05) numbers of
attaching bacteria hybridizing against probes Eub338, Erec482, and Ec. Similarly, increased
(P < 0.05) numbers of attaching bacteria hybridizing to probes Eub338 and Erec482 were
observed in ileal biopsies of CE dogs. Within the CE group, dogs with intestinal neoplasia had
the greatest (P < 0.05) number of attaching E. coli (Ec) and Enterobacteriaceae (Ebac1790) bacteria. Differences in the number of invasive bacteria within colonic mucosa included increased
total bacteria (Eub338) but decreased Bacteroides spp (Bac303) in IBD dogs relative to healthy
dogs; increased Enterobacteriaceae (Ebac1790) and E. coli (Ec) in GC dogs versus other CE dog
groups; and increased Bacteroides spp (Bac303) in dogs with neoplasia versus other CE dog
groups, P < 0.05 for all comparisons. In contrast, only E. coli (Ec) invasion (P < 0.05) was
observed in ileal biopsies of GC dogs.
Correlation between mucosal microbiota and inflammatory indices
The correlation between mucosal bacteria and inflammatory indices (CIBDAI score and histopathologic severity) was investigated to determine whether mucosal association might serve as
Table 3. Clinical characteristics of dogs studied. CIBDAI = canine IBD activity index with score reported at diagnosis.
Characteristic
IBD dogs
Neoplasia dogs
GC dogs
Controls
No. of females/no. of males
10/9
2/10
6/0
15/0
Mean age (yr.)
5.0
8.2
1.0
1.8
Mean CIBDAI score
7.4
5.1
3.5
0
Colitis
1
7
6
Enterocolitis
18
5
0
Dogs with signs of:
doi:10.1371/journal.pone.0147321.t003
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Mucosal Microbiota in Canine CE
Table 4. Number of ileal bacteria in healthy and diseased dogs.
Probe
A
IBD (n = 19)
B
Neoplasia (n = 12)
C
GC (n = 6)
D
Healthy (n = 15)
Eub338
Mean
8.9
18.7
14.6
12.2
Range
0–254
0–516
0–250
0–621
Mean
3.3 a
4.4 a
5.3 a
0.4
Range
0–115
0–115
0–77
0–20
Mean
0.8 a,b
7.2 a,b
2.0 a
0.7
Range
0–28
0–190
0–40
0–36
Mean
1.2 a
3.4
1.7 a
0.5
Range
0–44
0–160
0–35
0–36
Mean
3.4 a
6.8 a
8.7 a
0.6
Range
0–266
0–260
0–180
0–29
0
0.1 a
0
Erec482
Ebac1790
Ec
Bac303
Hel717
Mean
Range
0–3
1.9
0–149
Data expressed as mean and range.
a
b
significant (P < 0.05) difference between control and CE dogs.
significant (P < 0.05) difference between CE groups.
doi:10.1371/journal.pone.0147321.t004
a potential marker of disease activity in dogs with IBD and GC. Spearman’s rank correlation
coefficient showed no significant correlation between mucosal bacterial sub-populations in the
ileum and colon and the CIBDAI or histopathology score in IBD dogs and dogs with GC. However, there was a significant positive correlation (P = 0.026) between the number of total bacteria (probe Eub338) attached to the colonic mucosa and the CIBDAI score in IBD dogs.
Discussion
The pathogenesis of CE in dogs is believed to result from dysregulated host responses directed
against intestinal antigens, such as food antigens and bacteria [21–23]. In support of this
notion, culture-independent investigations using gene clone libraries, polymerase chain reaction (PCR), and DNA isolation for microbial abundance have demonstrated imbalance of the
microbiota in the diseased canine intestines [4–7,24]. Those data have largely been generated
only from IBD dogs using duodenal specimens for comparative analysis of bacterial populations. In regards to the evaluation of the distal intestinal microbiota detailed investigations are
clearly lacking. Only limited studies have incorporated FISH technology to investigate mucosal
microorganisms in colonic endoscopic biopsies of dogs with CE [10,11]. In the present study,
we report for the first time, the use of an array of FISH probes to visualize the composition and
spatial organization of canine ileal and colonic mucosal microbiota in health and disease. Our
results indicate that mucosal bacterial populations in the ileum and colon vary between different forms of CE in dogs.
Previous studies in humans have shown an association between GI signs, histopathologic
inflammation, and microbial imbalances in the intestine, especially with IBD (ie, Crohn’s
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Mucosal Microbiota in Canine CE
Table 5. Number of colonic bacteria in healthy and diseased dogs.
A
IBD (n = 19)
B
Neoplasia (n = 12)
C
GC (n = 6)
Mean
15.1
33.2 b
19.9 b
16.9
Range
0–815
0–2020
0–665
0–621
Mean
9.0 a,b
21.5 a,b
4.5 b
3.7
Range
0–397
0–1759
0–112
0–126
Mean
2.4 a,b
8.8 a,b
2.8 a,b
1.1
Range
0–140
0–623
0–53
0–51
Mean
2.8 a,b
7.0 a,b
1.9 a,b
0.5
Range
0–114
0–421
0–26
0–36
Mean
9.6 b
27.3 a,b
5.9 b
4.2
Range
0–1082
0–1872
0–210
0–225
Mean
0.4 a,b
0.1 a,b
0 a,b
Range
0–42
0–13
Probe
D
Healthy (n = 15)
Eub338
Erec482
Ebac1790
Ec
Bac303
Hel717
1.9
0–177
Data expressed as mean and range.
a
b
significant (P < 0.05) difference between control and CE dogs.
significant (P < 0.05) difference between CE groups.
doi:10.1371/journal.pone.0147321.t005
disease [CD] and ulcerative colitis [UC]) [25–28]. In general, these investigations have shown a
relative decrease in the bacterial phyla Firmicutes and Bacteroidetes, while an increase in Proteobacteria and Actinobacteria were observed with mucosal inflammation. Moreover, a reduction in the diversity of Clostridium clusters XIVa and IV (ie, Lachnospiraceae and C. coccoides
subgroups) are associated with IBD, suggesting that this bacterial group is important in disease
pathogenesis, possibly through their production of short chain fatty acids (SCFA) [29,30].
Mucosally invasive E. coli is an especially important bacterium that appears enriched in some
Crohn’s patients and linked to defects in innate immunity (ie, autophagy genes ATG16L1 and
IRGM) which can promote and perpetuate intestinal inflammation [31,32].
While earlier studies have predominantly reported changes in fecal microbiota, there is less
published information about the spatial organization of the mucosal microbiota seen with
human IBD. Knowledge of host-microbiota interactions, in particular the role of attaching and
invading bacteria, is important since an abnormal mucosal microbiota may interact more
closely with the innate immune system to modulate gut health and disease. For example, Kleessen et al used FISH to study differences in bacterial populations residing on the mucosal surface
and/or invading the terminal ileum and colonic tissues of human IBD patients [33]. Overall,
more bacteria were detected on the mucosal surface of IBD patients versus non-IBD controls.
Furthermore, bacterial invasion of the mucosa was evident in 83% of colonic specimens from
UC patients and in 56% of the ileal and 25% of the colonic specimens from CD patients. In
separate investigations, Swidsinski et al also used FISH evaluation of the mucosal microbiota
in biopsy specimens from humans with CE, including IBD [34,35]. Their results indicated
that IBD patients had high concentrations of mucosal bacteria and that an adherent mucosal
biofilm composed mainly of Bacteroides fragilis is a prominent feature of IBD. Common
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Fig 1. FISH of canine endoscopic biopsies. Triple color FISH identifies bacterial organisms within different
mucosal compartments of endoscopic ileal and colonic biopsies obtained from healthy dogs. Panel A = colon
biopsy hybridized with Cy3-Ebac1790; Panel B = colon biopsy hybridized with Cy3-Bac303; Panel C = ileum
hybridized with Cy3-Ebac1790; and Panel D = ileum hybridized with Cy3-Ec (E. coli). All other bacteria that
hybridize exclusively with the universal probe (Eub338-FITC) appear green. DAPI-stained colonic mucosa
with goblet cells appears blue. FM = free mucus; AM = adherent mucus.
doi:10.1371/journal.pone.0147321.g001
characteristics of all 3 studies included increased mucosal concentrations of Proteobacteria,
Enterobacteriaceae, and the Bacteroides/Prevotella cluster found in IBD patients.
The present study reveals that the ileal and colonic mucosal of healthy dogs and dogs with
CE is predominantly colonized by bacteria localized to the free and adherent mucus compartments. There were marked differences in the composition of the mucosal microbiota of healthy
versus CE dogs, with probes directed against Eubacterium rectale, Bacteroides, Enterobacteriaceae and E. coli significantly increased compared to these same bacterial sub-populations
observed in healthy dogs. The group of IBD dogs, in particular, had increased mucosal concentrations of Bacteroides and Enterobacteriaceae in both ileal and colonic biopsy specimens while
Clostridia and E. coli were increased in ileal and colonic mucus, respectively. An important
observation was the spatial re-distribution of Enterobacteriaceae and E. coli bacteria attached
to the surface epithelia and invasive within the mucosa of IBD dogs. This selective enrichment
of Enterobacteriaceae and E. coli in IBD tissues parallels reports of microbial shifts involving
the fecal and intestinal mucosal microbiota of other canine cohorts (as determined by 454-pyrosequencing, gene clone libraries), cats, and humans with IBD [4–7,13,26,29–31].
Dogs with granulomatous colitis (GC) had severe clinical disease in spite of relatively low
CIBDAI scores. This fact is related to the derivation of CIBDAI relative to abnormalities
involving colonic function alone; that is, only changes in stool character (ie, semi-solid feces,
mucus, and bright red blood) and frequency of defecation are observed most often [3]. The
concentrations of bacteria hybridizing with Eub338, Erec482, Ebac1790, and Ec probes were
significantly increased in the ileal and colonic mucus of GC dogs. All boxer dogs in this CE
group had increased numbers of attaching/invasive E. coli (AIEC) bacteria occurring as clusters
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Table 6. Spatial distribution of the number of ileal bacteria based on FISH.
Probe
Group
Eub338
Control
IBD
Neoplasia
GC
Erec482
Control
IBD
Neoplasia
GC
Ebac1790
Control
AM
SE
I
2.2
46.4
0.2
0.1
(0–67)
(0–621)
(0–16)
(0–2)
4.2b
31.1
0.4
0.1
(0–111)
(0–254)
(0–14)
(0–2)
17.3a,b
56
1.2a
0.1
(0–187)
(0–516)
(0–18)
(0–2)
0
44.3a,b
13
0.9
(0–250)
(0–71)
(0–20)
0.1
1.5
0.1
(0–15)
(0–20)
(0–4)
0
1.9a,b
11.2a
0.1
(0–69)
(0–115)
(0–9)
1.8a,b
15.1a
0.3a
0.1
(0–27)
(0–115)
(0–11)
(0–4)
16.2a,b
5.0
0
0
(0–77)
(0–49)
0
0
0
0.1
2.6
(0–2)
(0–36)
IBD
1.2a,b
1.9b
0.1b
0.1
(0–27)
(0–28)
(0–3)
(0–8)
Neoplasia
11.6a,b
16.5a,b
0.8b
0.1
(0–190)
(0–119)
(0–14)
(0–3)
4.5a
3.3
0
0
(0–40)
(0–32)
0
0
GC
Ec
FM
Control
0
1.9
(0–36)
0.6b
4.1
0.1b
0.1b
(0–23)
(0–44)
(0–3)
(0–3)
8.1b
5.2
0.3b
0.1
(0–160)
(0–40)
(0–4)
(0–3)
4.9b
1.6
0
0.4b
(0–35)
(0–17)
1.0
1.0
0.1
(0–28)
(0–29)
(0–6)
IBD
3.3a,b
10.0a
0.1
0.1
(0–77)
(0–266)
(0–8)
(0–3)
Neoplasia
13.0a,b
13.8a
0.4
0
(0–260)
(0–119)
(0–5)
26.3a,b
8.1a
0.2
(0–180)
(0–66)
(0–6)
7.8
0
0
0
0
0
0
IBD
Neoplasia
GC
Bac303
Control
GC
Hel717
Control
0
(0–10)
0
0
(0–149)
IBD
Neoplasia
0
0
0
0.4
a
(0–3)
(Continued)
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Table 6. (Continued)
Probe
Group
GC
FM
AM
SE
I
0
0
0
0
Data expressed as mean and range.
a
b
significant (P < 0.05) difference between control and CE dogs.
significant (P < 0.05) difference between CE groups.
doi:10.1371/journal.pone.0147321.t006
of organisms within inflamed ileal and colonic mucosa. Of interest is the previous finding that
the strains of E. coli isolated from Boxer dogs with GC have high phylogenetic resemblance to
E. coli associated with Crohn’s disease (ileitis) in humans [36]. Mutations in genes regulating
autophagy and bacterial clearance (ie, ATG16L1 and NCF2 genes) are thought to contribute to
enhanced host susceptibility to AIEC infection in humans and dogs, respectively [23,28,37].
Intestinal neoplasia involving the colorectal and ileal mucosa comprised a group of older
dogs with focal masses involving the descending colon and/or rectum (n = 7, all with adenocarcinoma) or having patchy or diffuse infiltrative disease involving the small and large intestines
and diagnosed with either adenocarcinoma (n = 2) or lymphoma (n = 3). In general, the intestinal tissues of cancer dogs contained the highest numbers of mucosal bacteria hybridizing to
probes Eub338, Erec482, Ebac1790, Ec, and Bac303 as compared to tissues obtained from
healthy dogs and dogs with non-cancer CE. This was especially true within the free/adherent
mucus compartments of both colonic and ileal biopsies. A similar trend was observed for
increased mucosal bacteria to be found attaching to surface epithelia or invading within colonic
(probes Eub338, Erec482, Ec and Bac303) and ileal (probes Ebac1790 and Ec) biopsies of cancer dogs versus dogs with other forms of CE.
The intestinal microbiota is increasingly linked with colorectal cancer (CRC) in humans.
Recent studies indicate that Fusobacterium spp. generate a pro-inflammatory microenvironment that is conducive to CRC progression likely through recruitment of tumor-infiltrating
immune cells [38]. Moreover, microbial mucosal shifts consisting of tumor enrichment with
both Fusobacterium nucleatum and Enterobacteriaceae have been observed in CRC tumors
[39,40], while invasive polymicrobial mucosal biofilms may serve as a distinct feature of more
proximal CRC in humans [41]. Whether increased mucosal concentrations of Fusobacteria
(not assessed in the present study) or other bacterial species are causally associated with mucosal inflammation and progression to CRC in dogs will require further study.
There was no statistical correlation between mucosal bacterial sub-populations in the ileum
and colon and the CIBDAI or histopathology score in IBD dogs and dogs with GC. This finding may have been due to the fact that we evaluated only the number of attaching/invading
bacteria, versus mucus-laden bacteria, in canine cohorts having mucosal inflammation. This
could also be influenced by the small study size (especially in GC dogs), the contribution from
non-bacterial factors mediating GI disease (ie, local immune response, motility disturbances),
and/or the difficulties inherent in the histopathologic interpretation of endoscopic biopsies
[42]. In spite of these factors, we did observe a significant positive correlation between the
number of total bacteria attached to the colonic mucosa and the CIBDAI score in IBD dogs
having moderate-to-severe disease severity.
There are some potential limitations in this study. It is possible that some dogs (n = 3) may
have received several days of antibiotics within 3 weeks of GI referral and diagnostic evaluation
for CE. In these instances, referring veterinarians had been treating animals short-term for routine non-GI related conditions including urinary tract infection (n = 1), pyoderma (n = 1), and
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Table 7. Spatial distribution of the number of colonic bacteria based on FISH.
Probe
Group
Eub338
Control
IBD
Neoplasia
GC
Erec482
Control
IBD
Neoplasia
GC
Ebac1790
Control
IBD
Neoplasia
GC
Ec
Control
IBD
Neoplasia
GC
Bac303
Control
IBD
Neoplasia
GC
Hel717
Control
IBD
Neoplasia
FM
AM
SE
I
10.1
55.4
2.0
0.2
(0–240)
(0–621)
(0–40)
(0–6)
22.5b
36.2
1.4
0.5a
(0–815)
(0–411)
(0–91)
(0–30)
91.2a,b
37.8
2.9a,b
0.8
(0–2020)
(0–516)
(0–49)
(0–14)
63.6a
14.7a
0.8b
0.3
(0–665)
(0–121)
(0–10)
(0–10)
4.5
9.8
0.5
0.1
(0–126)
(0–109)
(0–20)
(0–3)
8.2b
27.8a,b
0.4b
0.1
(0–397)
(0–270)
(0–20)
(0–8)
65.8a,b
18.8b
2.3a,b
0.2
(0–1759)
(0–320)
(0–27)
(0–6)
12.8b
4.9b
0.2b
0
(0–112)
(0–50)
(0–6)
0.4
4.2
0.1
(0–30)
(0–51)
(0–3)
0
4.1a,b
4.9
0.3a
0.1
(0–140)
(0–45)
(0–30)
(0–7)
29.2a,b
5.6
0.3a
0.1b
(0–623)
(0–49)
(0–14)
(0–2)
5.5a,b
5.0
0
0.5b
(0–53)
(0–26)
0.2
2.0
(0–9)
(0–36)
3.0a,b
8.0a
(0–100)
20.6a,b
(0–8)
0
0
0.1b
0.1
(0–114)
(0–3)
(0–1)
7.0a
0.4a,b
0.1b
(0–421)
(0–193)
(0–10)
(0–10)
1.6a,b
3.9
0.1
2.2b
(0–26)
(0–18)
(0–3)
(0–26)
8.4
6.8
1.4
0.2
(0–225)
(0–88)
(0–30)
(0–11)
22.1a,b
14.9
1.1
0.1a,b
(0–1082)
(0–163)
(0–55)
(0–3)
91.7a,b
14.9
2.2
0.4b
(0–1872)
(0–104)
(0–31)
(0–14)
16.3b
7.1
0
0
(0–210)
(0–56)
0
0
0
0
0
0
0.4
7.2
(0–30)
(0–177)
0
0a,b
0
a,b
0.3
(0–13)
(Continued)
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Table 7. (Continued)
Probe
Group
GC
FM
AM
SE
I
0
0
0
0
Data expressed as mean and range.
a
b
significant (P < 0.05) difference between control and CE dogs.
significant (P < 0.05) difference between CE groups.
doi:10.1371/journal.pone.0147321.t007
bacterial-mediated otitis externa (n = 1). Since FISH with rRNA-targeted probes is dependent
on the rRNA content (ie, metabolic activity) of individual bacteria, it is possible that previous
antibiotic administration may have reduced the proportion of some mucosal bacteria accessible
by FISH [35]. All dogs in the present study received routine colonic cleansing prior to collection of ileal and colonic mucosal biopsies. It is possible that dogs cleansed by colonic electrolyte
lavage and enemas might have had disrupted mucus compartments characterized by reduced
bacterial populations. However, we have previously investigated bacterial numbers by FISH in
pilot studies using untreated colonic specimens and found that mucus compartments do not
differ appreciably between purged vs. non-purged dogs (AEJ, unpublished observation). These
findings are in accordance with results in human IBD where differences in the mucus barrier
of intestinal bacteria were not observed between patients prepared by oral electrolyte lavage or
enema versus patients which did not receive colonic cleansing [43]. The precise mechanism(s)
Fig 2. FISH of canine endoscopic biopsies. Triple color FISH identifies bacterial organisms within different
mucosal compartments of endoscopic ileal and colonic biopsies obtained from dogs with CE. Panel A = colon
biopsy hybridized with Cy3-Erec482; Panel B = colon biopsy hybridized with Cy3-Ec (E. coli); Panel C = ileum
hybridized with Cy3-Ebac1790; and Panel D = ileum hybridized with Cy3-Bac303. All other bacteria that
hybridize exclusively with the universal probe (Eub338-FITC) appear green. DAPI-stained colonic mucosa
with goblet cells appears blue. AM = adherent mucus; SE = attached to surface epithelia; I = invasive within
mucosa.
doi:10.1371/journal.pone.0147321.g002
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
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Mucosal Microbiota in Canine CE
Fig 3. FISH of canine endoscopic biopsies. Triple color FISH identifies bacterial organisms present in biofilms adherent to the colonic epithelia in dogs with
inflammatory bowel disease (A), granulomatous colitis (B), and colorectal cancer (C). Bacteroides (Bac303-Cy3 probe–panel A), E. coli (Ec-Cy3 probe–panel
B), and Eubacterium rectale (Erec482-Cy3 –panel C) populations appear orange against green and blue backgrounds. All other bacteria that hybridize
exclusively with the universal probe (Eub338-FITC) appear green. DAPI-stained colonic mucosa with goblet cells appears blue.
doi:10.1371/journal.pone.0147321.g003
by which CE may alter the intestinal microbiota are beyond the scope of this study. It is possible that each of the disease conditions may have alterations in mucosal innate and adaptive
immune responses, particularly in release of REG-III, alpha and beta defensins or cathelcidins,
mucin production, increased intestinal permeability, and/or altered mucosal regulatory/cytotoxic T cell and dendritic cell activity [44]. It is also possible that the various canine CE affect
nutrient digestion and absorption, thereby altering the micronutrients available for the intestinal microflora.
Regarding the effect of formalin fixation [35] on integrity of the intestinal mucus layer (versus Carnoy’s fixative), our experiences using FISH in multiple species suggest that this is not a
problem and that the mucus layer remains largely intact even with routine tissue processing.
We have previously demonstrated the presence of an intact and largely continuous epithelial
mucus layer (via Alcian blue stain) in formalin-fixed endoscopic ileal and colonic specimens of
study dogs (data not shown). Moreover, other investigators have shown the utility of FISH
used on formalin-fixed biopsy specimens obtained from the GI tract of companion animals
[14,45] and humans [31,34,46–48].
Another potential factor impacting quantification of mucosal bacteria might be mechanical
artifacts associated with tissue processing (microtome cutting) and/or non-intended wash of
biopsy specimens by formalin during transport to the pathology laboratory [43]. Our previous
experiences have allowed us to readily identify these tissue artifacts (in companion animals and
mice) and to avoid these areas, if present, when performing mucosal bacterial counts. Finally,
gut microbial populations may potentially vary by age, gender, breed, and dietary consumption. Our own studies, evaluating the potential impact of age, body weight, and/or diet have
not identified any associations of microbial abundances with these variables in dogs to date
[5,49,50].
In summary, the present study indicates that dogs with CE exhibit imbalances in the composition and structure of their mucosal microbiota. Moreover, we show that mucosal bacterial
populations in the ileum and colon vary between the different forms of canine CE, and that
this is especially true for dogs with IBD or intestinal neoplasia where intestinal tissues are
selectively enriched in mucosal populations of Enterobacteriaceae and E. coli. These spatial,
segment-specific structure and differential response of select bacterial groups to intestinal
PLOS ONE | DOI:10.1371/journal.pone.0147321 February 3, 2016
15 / 18
Mucosal Microbiota in Canine CE
inflammation may be pivotal regarding the functional consequences of these alterations in the
pathogenesis of canine CE [49].
Author Contributions
Conceived and designed the experiments: AJ EC TA CM. Performed the experiments: AJ EC
RW TA SK CM MA. Analyzed the data: AJ CW YS MA. Contributed reagents/materials/analysis tools: AJ TA CM MA CW YS. Wrote the paper: AJ MA CM TA.
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